The proposed research investigates chromosome organization and function in bacteria and eukaryotic cells. Global whole-chromosome phenomena are examined by 4D time-lapse live cell imaging combined with genetic, molecular and biophysical approaches. These studies are united by the emerging concept that chromosomal events involve alternation of chromosomes between more and less constrained states, as we see in E.coli, yeast and mammalian cells. Elucidation of this fundamental pattern, and of consequences resulting from its perturbation, will provide new perspectives on chromosomal defects that underlie cancer and genetic diseases. E.coli research addresses: (i) the physical basis of bacterial chromosome (nucleoid) shape and state and its role in the mechanism by which bacterial sister chromosomes manage to segregate without a eukaryotic spindle apparatus; (ii) the mechanisms and roles of two dynamic behaviors - short time scale density oscillations and longer time scale extension/shortening cycles mediated by tethers; and (iii) the nature of coordination between chromosomal events and the cell division cycle, in which nucleoid state and dynamic behaviors are directly implicated. This work utilizes: (i) a platform for high throughput 4D (3D time lapse) fluorescence imaging and image analysis, with high spatial and temporal resolution, of the nucleoid, marked loci and protein complexes of interest, in living cells; (ii) genetic and optogenetic manipulations and perturbations; and (iii) whole chromosome numerical simulations of nucleoid organization. Eukaryotic chromosome research analogously examines chromosomes throughout the cell cycle, from the perspective of alternating compaction/expansion cycles. In mammalian cells, detailed studies will further investigate the just-discovered cycle in which individualized chromosome units first appear (at prophase) and progress to a different organizational state (at metaphase), and a subsequent cycle in which they prepare for sister segregation (Pre-anaphase). Of interest are three discovered features: a unique DNA state at prophase; inter-sister structural bridges at metaphase; and an abrupt jumping-apart of sisters at Pre-anaphase. Compaction/expansion events at other cell cycle stages will be analogously analyzed. This research involves high resolution 3D single cell time-lapse (4D) imaging, functional analysis via degron- and chemically-induced perturbations, and analysis of global chromosome dynamics by 4D tracking of fiduciary marks generated by fluorescent nucleotide incorporation. Parallel studies will be carried out in vegetatively growing yeast cells to assess functional requirements for compaction/expansion cycling, as we recently identified in that organism.